Ab46152

The samples were fixed in formalin and embedded in paraffin. Sections (thickness: 4 μm) were cut serially. The sections were heated in citrate buffer solution (Zsbio; Beijing, China) in a microwave for 20 minutes to antigen repaired. The sections were naturally cooled to room temperature before being treated with 3% H2O2 for 15 minutes to block endogenous peroxidase activity. Subsequently, they were incubated with the primary antibodies against anti-Nrf2 (1:200; ab62352; Abcam, Cambridge, MA, USA), anti-HO-1 (1:200; ab13248; Abcam, Cambridge, MA, USA), and anti-VEGF (1:200; ab46152; Abcam, Cambridge, MA, USA) overnight at 4°C and secondary antibodies for 1 h at 37°C. After staining with 3,3ʹ-diaminobenzidine (Zsbio; Beijing, China) and counterstaining with hematoxylin (Zsbio; Beijing, China), the sections were passed through graded ethanol and sealed. The Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA) was used to obtain the MOD value of the selected field. The microvessel density in tumors was assessed as suggested by Weidner.11 (link)

BGC-823 cells were cultured on coverslips. Cells were fixed with 4% paraformaldehyde (Zsbio; Beijing, China) for 15 min, permeated with 0.3% TritonX-100 (Solarbio; Beijing, China) for 20 min, and blocked with 10% goat serum (Zsbio; Beijing, China) for 30 min. Next, the cells were incubated with the primary antibodies against anti-Nrf2 (1:100; ab62352; Abcam, Cambridge, MA, USA), anti-HO-1 (1:100; ab13248; Abcam, Cambridge, MA, USA), and anti-VEGF (1:200; ab46152; Abcam, Cambridge, MA, USA) overnight at 4°C, and secondary antibodies at room temperature for 30 min. Cells were mounted on the slides with fluorescent mounting medium containing 4ʹ,6-diamidino-2-phenylindole (Zsbio; Beijing, China). The images were captured by fluorescence microscopy and analyzed using the Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA). The MOD was used to assess the expression levels.